Module organization and variance in protein-protein interaction networks

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1 Module organization and variance in protein-protein interaction networks Chun-Yu Lin 1, Tsai-Ling Lee 1, Yi-Yuan Chiu 1, Yi-Wei Lin 1, Yu-Shu Lo 1, Chih-Ta Lin 1, and Jinn-Moon Yang 1,2* 1 Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan 2 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan *Corresponding author Contact information: Address: 75 PO-Ai Street, Hsinchu, Taiwan, Tel: ext.56942; Fax: moon@faculty.nctu.edu.tw - 1 -

2 Supplementary Text 1: Homologous modules and human PPI network To observe topologies and functional similarities of homologous modules in module families, we collected 37,197 structural modules, from 187 reference modules across 1,442 organisms from the KEGG MODULE database 1. According to the data set, protein-aligned ratios of 96% (35,820) between homologous and their reference modules were more than 0.5 (Supplementary Fig. S1A). To determine topological similarity thresholds between reference and its homologous modules, we added intra-module PPIs using the following PPI databases: 1) 461,077 experimental PPIs from annotated PPI databases, including IntAct 2, BioGRID 3, DIP 4, MIPS 5, and MINT 6 ; 2) sequence-based homologous PPIs with joint E-values of among 461,077 experimental PPIs; and 3) 86,252 structure-based homologous PPIs with Z-scores of 4 8. Among 37,197 organism-specific structural modules, we added at least one PPI for 10,729 modules, and 80% PPI-aligned ratios between reference modules and their homologous modules were 0.3 (Supplementary Fig. S1B). Here, we set the protein-aligned ratio and PPI-aligned ratio to 0.5 and 0.3, respectively, to identify homologous modules of a module template

3 Supplementary Text 2: connectivity of modules A module is relatively autonomous and often has high connectivity (C t ) within a PPI network. To observe connectivity (C t ) of a module in a PPI network, we quantified the connectivity by C t m 9, C where n and m are the numbers of connected and PPIs in a module. A C t value of 1 indicates that are completely interconnected in a module. For C t of core (or ring) components, n and m are the numbers of connected core (or ring) and PPIs in a module. In this study, C t of core (or ring) components were evaluated while n is larger than 3. Here, we computed C t of modules using the human PPI network. Supplementary Fig. S2A shows the C t of core and ring components, module templates, and their respective extended modules. Extended modules were extended by onelayer of PPIs and in the module template (M). We assume that the module M consists of a set (P) of and a set (I) of protein-protein interactions (PPIs). The one-layer-extended module of this module M includes a set (P P ) of and a set (I ) of PPIs, where P consists of the n 2 interacting of each protein in set P; I consists of the PPIs of the in the set P P. Among 1,519 module templates, C t values of more than 0.6 were observed in 71% (1,081) of cases. In contrast, C t values were more than 0.6 for only 5% (71) of extended modules. Moreover, 90% of core components and 81% of ring components had C t values of 0.6. Similarly, 58,041 modules that were homologous to module templates had C t values of 0.6 in 76% of cases (44,319), whereas only 1% (842) of their extended modules had C t values of 0.6 (Supplementary Fig. S3A). These results indicate that core components have the highest connectivity, and that the modules also have high connectivity

4 Supplementary Text 3: biological functions of modules Through assembly and cooperation of in a PPI network, components of a module simultaneously perform certain biological functions. Based on the relative specificity similarity (RSS) 10 of GO terms, such as biological process (BP) and cellular component (CC), we computed AvgRSS scores to assess shared biological functions of all protein pairs in a module. The AvgRSS is defined as AvgRSS n n i 1 j 1 n RSS(i, j) C2, i j, where i and j are any two of a module and n is the number of in the module. To elucidate biological functions of modules, we compared module templates, their core and ring components, and their extended modules. For 1,519 module templates, BP and CC AvgRSS scores were more than 0.6 in 89% and 97% of cases, respectively (Supplementary Figs. S2B and S2C), and these scores were significantly higher than those of extended modules (Mann Whitney U test, P 0). In addition, BP and CC average AvgRSS scores of core components were higher than others, including ring components (Mann Whitney U test, P =2e-7 for BP; P =2e-21 for CC), whole module templates (P =1e-7 for BP; P =1e-14 for CC), and extended modules (P =3e-239 for BP; P =5e-262 for CC). CC AvgRSS scores (97%) of templates were slightly higher than those of their ring components (94%) with AvgRSS scores of 0.6. Furthermore, BP and CC AvgRSS scores were more than 0.6 for 81% and 94% of homologous modules, respectively (Supplementary Figs. S3B and S3C). Similarly, BP and CC average AvgRSS scores for core components of homologous modules were also significantly higher than those of ring components (P = for BP; P =3e-16 for CC). For example, BP and CC AvgRSS scores for the CDC2 PCNA CCNB1 GADD45B homologous module in H. sapiens were 0.79 and 0.84, but for extended modules they were only 0.43 and 0.25, respectively. The core components of this module had high BP and CC AvgRSS scores of 0.89 and 0.85, respectively. These results indicate that homologous modules of a template have highly similar biological functions and that their core components regulate similar biological processes and are often localized to the same cellular compartment

5 Supplementary Text 4: GO term analysis of essential GO terms provide the descriptions of the biological process (BP), cellular component (CC), and molecular function (MF) of a protein 11. According to a modified term frequency-inverse document frequency (TF-IDF) scoring scheme 12, we identified 160 essential MF terms that describe the functional relationships of essential and core of the module families (Supplementary Table S1). First, we collected 8,364 essential, called EP8364, from the DEG database and 160,598, called CG27, over 27 completed genomes. The in these two sets contained at least one GO MF or GO BP terms. The "occurrence ratio" (CR t ) of a GO MF term (t) was defined as CR t = P t /T, where P t is the number of with term t, and T is the total number of in the given set. For example, the occurrence ratio of the term "rrna binding" was in the EP8364 set for P t = 416 and T = 8,364. The distribution of the occurrence ratios of 1,886 GO MF terms between core and essential was similar (Pearson's r = 0.86). In contrast, Pearson's r for 4,699 GO BP terms was 0.27, because BP terms often describe a series of events accomplished by one or more ordered assemblies of molecular functions. The MF and BP terms are suitable for analyses at the protein and module levels, respectively. Next, we developed a "unique ratio" (UR) to statistically measure the importance of the GO MF term (i.e., specificity of a protein) by a modified TF-IDF scoring scheme 12. The unique ratio of a GO MF term t was defined as UR t = CR EP t /CR CG t, where CR EP t and CR CG t are the occurrence ratios of term t in sets EP8364 and CG27, respectively. For example, the unique ratio of the term "rrna binding" was determined as 9.72 for CR EP t = and CR CG t = Finally, we selected 160 essential GO MF terms that are statistically significant specificity to essential with UR 2 and p-value 0.05 (hypergeometric distribution). We discarded the terms of specific species (e.g., "azobenzene reductase activity") and those with high usage but without the specificity (e.g., "protein binding"). To analyze the characteristics and functions of the core components, we clustered these 160 essential GO MF terms into 12 groups: Translation (30 terms, 17%), Transcription (9 terms, 5%), (22 terms, 12%), Lipid (11 terms, 6%), Amino acid (10 terms, 6%), DNA replication (11 terms, 6%), RNA degradation (6 terms, 3%), Purine (11 terms, 6%), Pyrimidine (4 terms, 2%), Cell cycle (4 terms, 2%), Oxidative phosphorylation (5 terms, 3%) (Supplementary Fig. S4A and Table S1). The largest percentage (17%) of the essential GO MF terms was assigned to Translation, including such terms as "rrna binding" (UR = 9.72), "translation release factor activity, codon specific" (UR = 6.48), - 5 -

6 "structural constituent of ribosome" (UR = 4.72), and "trna binding" (UR = 8.38). In the process of transcription, the information contained in a segment of DNA is transferred to a newly assembled piece of mrna. The central dogma of molecular biology, including DNA replication, transcription, and translation, is the fundamental of life for sequence information transfer 13. Among the 160 essential GO MF terms, 31% of essential GO MF terms were involved in the central dogma (Supplementary Fig. S4A). Furthermore, we also analyzed the percentage of GO MF groups in 3,366 essential (Supplementary Fig. S4B). Seventy-two percent of the essential were annotated with GO MF terms that were related to the central dogma, such as "translation" (55%). Among the 160 essential GO MF terms, 33 terms (21%; e.g., "acetyl-coa carboxylase activity", UR = 9.25) were recorded for and Lipid s, which mediate the energy balance of organisms and constitute various biochemical processes responsible for the formation, breakdown, and interconversion 14,15. Further, 16 essential GO MF terms were included in Amino acid (e.g., "cysteine desulfurase activity", UR = 6.89) and RNA degradation (e.g., "3-5 exonuclease activity", UR = 5.27), which play an important role in energy balance through the reuse of RNA and amino acids. Purine (e.g., "ATP-dependent RNA helicase activity", UR = 5.04) and Pyrimidine (e.g., "thymidylate kinase activity", UR = 6.98) s are regarded as modular minimal cell model 16. Generation of biological energy occurs mainly through the pathways contained in the Oxidative phosphorylation group 17. These results demonstrate that a majority of these 160 essential GO MF terms are indispensable for the survival of an organism

7 Supplementary Text 5: Microarray expression data sets of 9 tumor types To identify genes with significant expression change between tumor and corresponding normal tissues, we collected 6 gene expression data sets, including 9 different tumor types, from GEO 18. Each expression data set comprising 3 tumor samples and corresponding normal samples were obtained using the most comprehensive human expression array platform (HG U133 Plus 2.0; Supplementary Table S3). For each GeneChip array that passed quality control checks, quantile normalization was evaluated and gene expression values were calculated based on the log 2 scale using the RMA algorithm 19. Next, the Bioconductor method limma 20, based on a modified t-statistic, was utilized to measure the fold change value (FC) of each gene between tumor samples and corresponding normal samples. For each gene g, the FC is defined as FC g Average log Average N Tg 2 2 log g, where T g and N g is the expression in the t tumor samples (T 1, T 2,,T t ) and n corresponding normal samples (N 1, N 2,,N n ). The adjusted P-values were applied for multiple hypothesis testing using Benjamini and Hochberg s method 21 and the False Discovery Rate (FDR) was controlled at 5%. Finally, the genes with adjusted P-value <0.05 and fold change >1.3 are considered as significantly changed genes

8 Percentage of modules Percentage of modules A B 100% 98% 96% 94% 92% 90% 88% 86% 84% 82% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Protein-aligned ratio PPI-aligned ratio Supplementary Figure S1. Evaluations of topological similarity. (A) The distribution of protein-aligned ratios and their fractions of 37,197 KEGG organism-specific structural complexes. (B) The distribution of PPI-aligned ratios and their fractions of 10,729 KEGG structural complexes, with intra-module interactions added using three PPI databases

9 Percentages Percentage Percentage A 70% 60% 50% 40% 30% 20% Core Ring Module templates Extended modules B 70% 60% 50% 40% 30% 20% Core Ring Module templates Extended modules Biological Processes 10% 10% 0% 0% C Connectivity Cellular Components Average RSS score 70% 60% 50% 40% 30% 20% Core Ring Module templates Extended modules 10% 0% Average RSS score Supplementary Figure S2. Distributions of connectivity (C t ) and average relative specificity similarity (AvgRSS) across core components and ring components of module templates and their extended modules. (A) C t distributions of core and ring components, module templates, and extended modules using 1,519 module templates; the extended module is a sub-network that includes one-layer extensions of PPIs and of the module template. Distributions of AvgRSS scores of GO (B) biological processes and (C) cellular components for core and ring components, module templates, and extended modules

10 Percentages Percentage Percentages A 70% 60% 50% 40% 30% Core Ring Homologous modules Extended modules B 70% 60% 50% 40% 30% Biological Processes Core Ring Homologous modules Extended modules 20% 20% 10% 10% 0% 0% C Connectivity Cellular Components Average RSS score 70% 60% 50% 40% Core Ring Homologous modules Extended modules 30% 20% 10% 0% Average RSS score Supplementary Figure S3. Connectivity (C t ) distributions and AvgRSS score distributions of GO Biological processes (BP) and Cellular Components (CCs) across core components and ring components of homologous modules and their extended modules. (A) C t distributions, (B) GO BP AvgRSS score distribution, and (C) GO CC AvgRSS score distributions of core components and ring components of 58,041 homologous modules and their respective extended modules

11 Translation lipid Transcription Purine DNA replication Amino acid RNA degradation Cell cycle Oxidative phosphorylation Pyrimidine Others Percentage of A Pyrimidine 2% Oxidative phosphorylation 3% Cell cycle 2% RNA degradation 4% B 60% 50% 40% Amino acid 6% Others 23% DNA replication 7% Translation 19% Purine Transcription 6% 7% 14% Lipid 7% Essential Core component (Interface evolution score 7) Ring component (Interface evolution score < 7) 30% 20% 10% 0% GO MF groups Supplementary Figure S4. Gene ontology (GO) molecular function (MF) terms of essential, core, and ring. (A) 12 groups of 160 essential GO MF terms taken from 8,364 essential were sampled based on KEGG pathways and GO; (B) Percentages of GO MF groups in 3,366 essential (blue), 379 core (red), and 339 ring (green)

12 sequence-specific enhancer binding RNA polymerase II transcription elongation regulator activity transcription termination factor activity mrna binding DNA-directed RNA polymerase activity DNA topoisomerase (ATP-hydrolyzing) activity general RNA polymerase II transcription factor activity sigma factor activity ribonucleoside binding DNA replication origin binding DNA clamp loader activity single-stranded DNA binding DNA-directed DNA polymerase activity DNA primase activity DNA topoisomerase type I activity ATP-dependent DNA helicase activity cytidylate kinase activity phosphatidate cytidylyltransferase activity four-way junction helicase activity DNA helicase activity trna binding structural constituent of ribosome translation initiation factor activity translation elongation factor activity aminoacyl-trna hydrolase activity ribonuclease P activity alanine-trna ligase activity arginine-trna ligase activity aspartate-trna ligase activity asparagine-trna ligase activity cysteine-trna ligase activity glutamate-trna ligase activity glycine-trna ligase activity histidine-trna ligase activity isoleucine-trna ligase activity leucine-trna ligase activity lysine-trna ligase activity methionine-trna ligase activity phenylalanine-trna ligase activity proline-trna ligase activity serine-trna ligase activity threonine-trna ligase activity tryptophan-trna ligase activity tyrosine-trna ligase activity valine-trna ligase activity 5S rrna binding poly(a) RNA binding translation release factor activity, codon specific rrna binding ribosome binding 3'-5'-exoribonuclease activity nuclease activity ribonuclease III activity ribonuclease activity 3'-5' exonuclease activity 5'-3' exonuclease activity chromatin binding cyclin-dependent protein kinase inhibitor activity frizzled binding Wnt-protein binding 6-phosphofructokinase activity UTP:glucose-1-phosphate uridylyltransferase activity dihydrolipoyl dehydrogenase activity fructose-bisphosphate aldolase activity glucose-6-phosphate isomerase activity glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) methenyltetrahydrofolate cyclohydrolase activity methylenetetrahydrofolate dehydrogenase (NADP+) activity dolichyl-diphosphooligosaccharide-protein glycotransferase phosphomannomutase activity phosphoglycerate kinase activity phosphoglycerate mutase activity phosphopyruvate hydratase activity pyruvate dehydrogenase (acetyl-transferring) activity dihydrolipoyllysine-residue acetyltransferase activity pyruvate kinase activity ribulose-phosphate 3-epimerase activity ribose-5-phosphate isomerase activity transketolase activity Essential Core component (Interface evolution score 7) Ring component (Interface evolution score < 7) 0 Essential GO MF terms Supplementary Figure S5. s of 160 essential GO MF terms between essential, core, and ring s of each set are only labeled with the significant enrichment, as determined by p-values of 0.05 (hypergeometric distribution) in each GO term

13 triose-phosphate isomerase activity phosphatidylinositol N-acetylglucosaminyltransferase activity NADP or NADPH binding CTP synthase activity thioredoxin-disulfide reductase activity thymidylate kinase activity thymidylate synthase activity GMP synthase (glutamine-hydrolyzing) activity IMP dehydrogenase activity adenine phosphoribosyltransferase activity ATP-dependent RNA helicase activity adenylate kinase activity guanylate kinase activity hypoxanthine phosphoribosyltransferase activity phosphoribosylaminoimidazolesuccinocarboxamide synthase ribose phosphate diphosphokinase activity ATPase activity nucleoside-triphosphatase activity mannosyltransferase activity 1-acylglycerol-3-phosphate O-acyltransferase activity acetyl-coa carboxylase activity biotin-[acetyl-coa-carboxylase] ligase activity [acyl-carrier-protein] S-malonyltransferase activity 3-oxoacyl-[acyl-carrier-protein] synthase activity 3-oxoacyl-[acyl-carrier-protein] reductase activity glycerol-3-phosphate O-acyltransferase activity phosphatidylserine decarboxylase activity prenyltransferase activity transferase activity, transferring pentosyl groups inorganic diphosphatase activity hydrogen-exporting ATPase activity, phosphorylative mechanism hydrogen ion transporting ATP synthase activity, rotational proton-transporting ATPase activity, rotational mechanism 3 iron, 4 sulfur cluster binding 3-isopropylmalate dehydratase activity DNA (cytosine-5-)-methyltransferase activity aspartate kinase activity glutamine-fructose-6-phosphate transaminase (isomerizing) glycine hydroxymethyltransferase activity methionine adenosyltransferase activity shikimate 5-dehydrogenase activity shikimate kinase activity tryptophan synthase activity cysteine desulfurase activity acyl carrier activity magnesium ion binding adenyl-nucleotide exchange factor activity NAD+ kinase activity dephospho-coa kinase activity dihydrofolate reductase activity tetrahydrofolylpolyglutamate synthase activity nicotinate-nucleotide diphosphorylase (carboxylating) activity pantothenate kinase activity receptor signaling protein tyrosine kinase activity riboflavin synthase activity ribonucleoside-diphosphate reductase activity structural constituent of cytoskeleton enzyme activator activity 7S RNA binding penicillin binding holo-[acyl-carrier-protein] synthase activity biotin binding excinuclease ABC activity cobalt ion transmembrane transporter activity P-P-bond-hydrolysis-driven protein transmembrane transporter oxidoreductase activity, acting on the aldehyde or oxo group of oxidoreductase activity, acting on paired donors, with sulfurtransferase activity hydrolase activity, acting on acid anhydrides, catalyzing hydro-lyase activity carbon-nitrogen ligase activity, with glutamine as amido-n-donor structural constituent of nuclear pore snorna binding potassium ion binding phosphopantetheine binding peptide deformylase activity identical protein binding SMAD binding transition metal ion binding NADH dehydrogenase (quinone) activity Essential Core component (Interface evolution score 7) Ring component (Interface evolution score < 7) 0 Essential GO MF terms Supplementary Figure S5. s of 160 essential GO MF terms between essential, core, and ring s of each set are only labeled with the significant enrichment, as determined by p-values of 0.05 (hypergeometric distribution) in each GO term. (Continued)

14 Percentage of modules 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Ring-only Core-ring Core-only Module size Supplementary Figure S6. Module size distributions against percentages of modules with all belonging to core (core-only, red), ring (ring-only, blue), or both (core ring, green)

15 1,160 profiles 1,085 profiles CDK1-CCNB1 CDK1-PCNA PCNA-CCNB1 GADD45B-PCNA GADD45B-CCNB1 GADD45B-CDK1 CDK1-CCNB1 CDK1-PCNA PCNA-CCNB1 GADD45B-PCNA GADD45B-CCNB1 GADD45B-CDK1 Genes Expression value A Collect 7,208 gene expression data sets ( 3 samples) from the GEO database. B (1) E j E all (2) S j S all Select the gene j in a gene expression set based on the following criteria: average expression ( ) mean expressions of all genes ( ) in a gene expression set; or the standard deviation of expressions (S j ) the standard deviation of expression values for all genes (S all ) in the gene expression set. Collect expression profiles with expression values of all in a module. C Samples CDK1 PCNA CCNB1 GADD45B Expression value Low High PCNA CCNB1 CDK1 GADD45B Calculate Pearson's r values for each PPI within the module to construct correlation matrix. Select profiles containing at least one PPI of the module with high Pearson s r h (here, h was set at 0.3, 0.5, or 0.7) (assumed as an active module performing biological functions in a cell). GSE10061 GSE10070 GSE10088 GSE10096 GSE1010 GSE10172 GSE10096 GSE1010 GSE10061 GSE10070 GSE10088 GSE10172 Pearson's r 0.5 Pearson's r < profiles Co-expression ratio of CDK1-CCNB1 = 0.76 Evaluate the co-expression ratio (CE) of each PPI within the module at the threshold h Supplementary Figure S7. Gene co-expressions of core and ring PPIs in the modules using 7,208 H. sapiens gene sets from Gene Expression Omnibus (GEO). (A) The main procedure for collecting gene profiles and evaluating co-expression of core and ring PPIs in modules. (B) Gene expression profiles are collected by discarding non-significant genes with low expression and low expression variance. (C) Co-expression profiles of all protein pairs (PPIs) from the CDK1 PCNA CCNB1 GADD45B module

16 Average percentage of % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Ring Core Module organizational variance Supplementary Figure S8. The relationship between module organizational variance and core/ring composition of modules

17 Supplementary Figure S9. The boxplot of protein functional variance for ring and core

18 RFC2-RFC5 RFC4-RFC5 RFC4-RFC2 RFC5-RFC3 RFC4-RFC3 RFC2-RFC3 RAD17-RFC4 RAD17-RFC3 RAD17-RFC5 RAD17-RFC2 RAD17-RAD17 RAD17-HUS1 HUS1-RAD17 RAD17-RAD9A RAD17-RAD9A HUS1-RAD9A Co-expression ratio 127 species (5 taxonomic division) RFC4-RFC5 RFC2-RFC5 RFC4-RFC2 RFC2-RAD9A RFC4-RAD9A RFC5-RAD9A RAD17-RFC5 RAD17-RFC2 RAD17-RFC4 RAD17-RAD9A RAD17-RFC3 HUS1-RAD17 HUS1-RFC3 RAD1-RAD17 RAD1-HUS1 RAD1-RFC3 A B RAD17-RFC checkpoint module (Homo sapiens) HUS1 RFC2 RFC3 RFC5 RAD17 RFC4 RAD1 RAD9A PPI similarity (Join E-value) D Network topology HUS1 4 RAD9A RFC3 RAD RFC2 4 RAD1 Degree 17 RFC4 13 RFC5 E Essentiality HUS1 RFC3 RAD17 RAD9A RFC2 RAD RFC5 RFC4 Mapped essential protein C RFC2 Core components HUS1 RFC3 4.4 RAD17 RAD9A RAD1 10 RFC5 10 RFC N/A Ring components PPI evolution score F G Co-expression Co-expression ratio of RFC2-RFC5= 0.74 Core Ring Module variances RFC2-5 module HUS1 MOV (MOV= 1.00) 0.33 = 0.71 RFC3 RAD RAD9A RFC2 RAD1 PFV=1 1 1 RFC4 RAD17-RFC RFC5 module (MOV= 0.93) Supplementary Figure S10. Characteristics and variance of core and ring components of RAD17 RFC checkpoint module. (A) The RAD17 RFC checkpoint module. (B) The module family profile includes 8 and 16 PPI families. (C) Solid circles and lines denote the 5 core and 10 core PPIs, respectively, and dashed circles and lines denote the 3 ring and 6 ring PPIs, respectively. The PPI evolution scores are indicated. (D) Degrees of core and ring in the human PPI network, including 2,391 and 11,181 PPIs. (E) Orange circles indicate mapped essential. (F) Co-expression ratios of 16 PPIs among 309 expression profiles selected from 7,208 gene sets. (G) The RAD17 RFC supermodule comprises three modules with protein/module variance, including RFC2 5 (green), RAD17 RFC (pink), and RAD17 RFC The solid circle and line denote the core protein and PPI, respectively. The dash circle and line indicate the ring protein and PPI, respectively

19 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 GO: rrna binding Translation E GO: glycine-trna ligase activity Translation E GO: trna binding Translation E GO: glutamate-trna ligase activity Translation E GO: proline-trna ligase activity Translation E GO: valine-trna ligase activity Translation E GO: methionine-trna ligase activity Translation E GO: arginine-trna ligase activity Translation E GO: lysine-trna ligase activity Translation E GO: phenylalanine-trna ligase activity Translation E GO: leucine-trna ligase activity Translation E GO: translation release factor activity, codon specific Translation E GO: isoleucine-trna ligase activity Translation E GO: tyrosine-trna ligase activity Translation E GO: cysteine-trna ligase activity Translation E GO: ribonuclease P activity Translation E GO: threonine-trna ligase activity Translation E GO: asparagine-trna ligase activity Translation E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database. ratio (IES 7)

20 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1204 GO: serine-trna ligase activity Translation E GO: alanine-trna ligase activity Translation E GO: histidine-trna ligase activity Translation E GO: aspartate-trna ligase activity Translation E GO: S rrna binding Translation E GO: tryptophan-trna ligase activity Translation E GO: structural constituent of ribosome Translation E GO: aminoacyl-trna hydrolase activity Translation E GO: poly(a) RNA binding Translation E GO: ribosome binding Translation E GO: translation elongation factor activity Translation E GO: translation initiation factor activity Translation E GO: triose-phosphate isomerase activity E GO: ribose-5-phosphate isomerase activity E GO: dihydrolipoyl dehydrogenase activity E GO: phosphoglycerate kinase activity E GO: dihydrolipoyllysine-residue acetyltransferase activity E GO: methenyltetrahydrofolate cyclohydrolase activity E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database. ratio (IES 7)

21 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification GO: GO: GO: GO: GO: GO: GO: GO: GO: methylenetetrahydrofolate dehydrogenase (NADP+) activity transketolase activity phosphopyruvate hydratase activity glucose-6-phosphate isomerase activity UTP:glucose-1-phosphate uridylyltransferase activity phosphomannomutase activity ribulose-phosphate 3-epimerase activity glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) activity phosphatidylinositol N- acetylglucosaminyltransferase activity pyruvate dehydrogenase (acetyl-transferring) activity (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) E E E E E E E E E GO: GO: phosphoglycerate mutase activity GO: fructose-bisphosphate aldolase activity dolichyl-diphosphooligosaccharide-protein GO: glycotransferase activity GO: phosphofructokinase activity GO: NADP or NADPH binding a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database E E E E E E

22 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: pyruvate kinase activity acetyl-coa carboxylase activity lipid [acyl-carrier-protein] S-malonyltransferase activity lipid 3-oxoacyl-[acyl-carrier-protein] synthase activity lipid 3-oxoacyl-[acyl-carrier-protein] reductase activity lipid prenyltransferase activity lipid biotin-[acetyl-coa-carboxylase] ligase activity lipid phosphatidylserine decarboxylase activity lipid 1-acylglycerol-3-phosphate O-acyltransferase activity lipid glycerol-3-phosphate O-acyltransferase activity lipid mannosyltransferase activity lipid transferase activity, transferring pentosyl groups lipid (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) E E E E E E E E E E E E GO: DNA topoisomerase (ATP-hydrolyzing) activity Transcription E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database

23 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification GO: general RNA polymerase II transcription factor activity (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) Transcription E GO: transcription termination factor activity Transcription E GO: ribonucleoside binding Transcription E GO: sigma factor activity Transcription E GO: transcription elongation regulator activity Transcription E GO: DNA-directed RNA polymerase activity Transcription E GO: mrna binding Transcription E GO: sequence-specific enhancer binding RNA polymerase II transcription factor activity Transcription E GO: ATP-dependent RNA helicase activity Purine E GO: GMP synthase (glutamine-hydrolyzing) activity Purine E GO: guanylate kinase activity Purine E GO: adenine phosphoribosyltransferase activity Purine E GO: adenylate kinase activity Purine E GO: phosphoribosylaminoimidazolesuccinocarboxamide Purine synthase activity E GO: ribose phosphate diphosphokinase activity Purine E GO: IMP dehydrogenase activity Purine E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database

24 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c GO: hypoxanthine phosphoribosyltransferase activity Purine GO: ATPase activity Purine GO: nucleoside-triphosphatase activity Purine GO: DNA replication origin binding DNA replication GO: cytidylate kinase activity DNA replication GO: phosphatidate cytidylyltransferase activity DNA replication GO: DNA primase activity DNA replication GO: four-way junction helicase activity DNA replication GO: DNA helicase activity DNA replication GO: DNA-directed DNA polymerase activity DNA replication GO: DNA topoisomerase type I activity DNA replication GO: DNA clamp loader activity DNA replication GO: single-stranded DNA binding DNA replication GO: ATP-dependent DNA helicase activity DNA replication GO: cysteine desulfurase activity Amino acid GO: methionine adenosyltransferase activity Amino acid a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database. Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) E E E E E E E E E E E E E E E E

25 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: GO: shikimate 5-dehydrogenase activity tryptophan synthase activity glutamine-fructose-6-phosphate transaminase (isomerizing) activity DNA (cytosine-5-)-methyltransferase activity glycine hydroxymethyltransferase activity 3-isopropylmalate dehydratase activity aspartate kinase activity shikimate kinase activity 3'-5' exonuclease activity 3'-5'-exoribonuclease activity ribonuclease activity ribonuclease III activity 5'-3' exonuclease activity nuclease activity Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid RNA degradation RNA degradation RNA degradation RNA degradation RNA degradation RNA degradation (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) E E E E E E E E E E E E E E GO: frizzled binding Cell cycle E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database

26 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 GO: cyclin-dependent protein kinase inhibitor activity Cell cycle E GO: Wnt-protein binding Cell cycle E GO: chromatin binding Cell cycle E ratio (IES 7) Oxidative GO: inorganic diphosphatase activity phosphorylation E Oxidative GO: iron, 4 sulfur cluster binding phosphorylation E GO: hydrogen ion transporting ATP synthase activity, Oxidative rotational mechanism phosphorylation E GO: proton-transporting ATPase activity, rotational Oxidative mechanism phosphorylation E GO: hydrogen-exporting ATPase activity, Oxidative phosphorylative mechanism phosphorylation E Pyrimidine GO: thymidylate kinase activity E Pyrimidine GO: thymidylate synthase activity E Pyrimidine GO: CTP synthase activity E Pyrimidine GO: thioredoxin-disulfide reductase activity E GO: snorna binding Others E GO: peptide deformylase activity Others E GO: dihydrofolate reductase activity Others E GO: riboflavin synthase activity Others E GO: ribonucleoside-diphosphate reductase activity Others E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database

27 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 GO: dephospho-coa kinase activity Others E GO: holo-[acyl-carrier-protein] synthase activity Others E GO: adenyl-nucleotide exchange factor activity Others E GO: P-P-bond-hydrolysis-driven protein transmembrane transporter activity ratio (IES 7) Others E GO: hydro-lyase activity Others E GO: sulfurtransferase activity Others E GO: pantothenate kinase activity Others E GO: oxidoreductase activity, acting on the aldehyde or oxo group of donors, disulfide as acceptor Others E GO: S RNA binding Others E GO: penicillin binding Others E GO: biotin binding Others E GO: oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of oxygen Others E GO: transition metal ion binding Others E GO: NAD+ kinase activity Others E GO: hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances Others E GO: chaperone binding Others E carbon-nitrogen ligase activity, with glutamine as GO: Others E amido-n-donor a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 7 and at least one GO MF term annotation in GO database

28 Supplementary Table S1. The 160 essential GO molecular function (MF) terms. (Continued) GO ID GO term Classification (27 species genomes a ) b (27 species genomes; total 160,598 Number of essential ; total 8,364 ratio c Hypergeometric P-value in templates (IES 7) d Occurrence ratio (IES 7; total 1,204 ratio (IES 7) GO: receptor signaling protein tyrosine kinase activity Others E GO: excinuclease ABC activity Others E GO: SMAD binding Others E GO: NADH dehydrogenase (quinone) activity Others E GO: structural constituent of cytoskeleton Others E GO: structural constituent of nuclear pore Others E GO: cobalt ion transmembrane transporter activity Others E GO: tetrahydrofolylpolyglutamate synthase activity Others E GO: enzyme activator activity Others E GO: nicotinate-nucleotide diphosphorylase (carboxylating) activity Others E GO: phosphopantetheine binding Others E GO: magnesium ion binding Others E GO: potassium ion binding Others E GO: identical protein binding Others E GO: acyl carrier activity Others E a The 27 species genome set (160,598 annotated 1 GO MF terms) consists of 25 species in DEG and 2 species in module template set. b The occurrence ratio of a GO MF term is defined as the number of annotated this terms divided by the total number of in the set. c The unique ratio of a GO MF term is defined as the occurrence ratio of a GO MF term divided by the occurrence ratio in 27 species genome set. d The of module templates represent the core component in module families with interface evolution score (IES) 8 and at least one GO MF term annotation in GO database